This invention is in the field of plant molecular biology. This invention pertains to nucleic acid fragments from plants encoding proteins that are homologs of the undecaprenyl diphosphate and dehydrodolichyl diphosphate synthases (cis-prenyltransferases) previously identified only in microbes. More specifically, this invention pertains to homologs from wheat, grape, soybean, rice, African daisy, rubber tree and pot marigold.
Plants synthesize a variety of hydrocarbons built up of isoprene units (C5H8), termed polyisoprenoids (Tanaka, Y. In Rubber and Related Polyprenols. Methods in Plant Biochemistry; Dey, P. M. and Harborne, J. B., Eds., Academic Press: San Diego, 1991; Vol. 7, pp 519-536). Those with from 45 to 115 carbon atoms, and varying numbers of cis- and trans- (Z- and E-) double bonds, are termed polyprenols, while those of longer chain length are termed rubbers (Tanaka, Y. In Minor Classes of Terpenoids. Methods in Plant Biochemistry; Dey, P. M. and Harborne, J. B., Eds., Academic Press: San Diego, 1991; Vol. 7, pp 537-542). The synthesis of these compounds is carried out by a family of enzymes termed prenyltransferases, which catalyze the sequential addition of C5 units to an initiator molecule.
The initiator molecules themselves are derived from isoprene units through the action of distinct prenyltransferases, and are allylic terpenoid diphosphates such as dimethylallyldiphosphate (DMAPP), but more usually the C10 compound geranyl diphosphate (GPP), the C15 compound famesyl diphosphate (FPP) or the C20 compound geranylgeranyl diphosphate (GGPP). Genes encoding the enzymes which synthesize these allylic terpenoid diphosphates have been cloned from a number of organisms, including plants, and all of these genes encode polypeptides with conserved regions of homology (McGarvey et al., Plant Cell 7:1015-1026 (1995); Chappell, J., Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:521-547 (1995)). All of these gene products condense isoprene units in the trans-configuration. Prenyltransferases which condense isoprene units in a cis-configuration have not been identified in higher animals or plants, nor have prenyltransferases catalyzing extension of the polyisoprenoid chain beyond the C20 compound geranylgeranyl diphosphate.
A gene encoding octaprenyl diphosphate (OPP) synthase from the bacterium E. coli was identified (Asai et al., Biochem. Biophys. Res. Commun. 202:340-345 (1994)), and more recently, genes encoding bacterial undecaprenyl diphosphate (UPP) synthases (Shimizu et al., J. Biol. Chem. 273:19476-19481 (1998); Apfel et al., J. Bacteriol. 181:483-492 (1999)) and yeast dehydrodolichyl diphosphate (Dedol-PP) synthase (Sato et al., Mol. Cell. Biol. 19:471-483 (1999)) were identified. OPP synthase generates the all-trans polyisoprenoid side chain of biological quinones (ubiquinone-8, menaquinone-8 and dimethylmenaquinone-8), and its primary structure contains regions of similarity with GPP, FPP and GGPP synthases. UPP synthase and Dedol-PP synthase generate cis-polyisoprenoids, and their primary structures are related to each other but distinct from those of OPP, GPP, FPP and GGPP synthases.
There are several suggested functions for plant polyisoprenoids. Terpenoid quinones are most likely involved in photophosphorylation and respiratory chain phosphorylation. Rubbers have been implicated in plant defense against herbivory, possibly serving to repel and entrap insects and seal wounds in a manner analogous to plant resins. The specific roles of the C45-C115 polyprenols remain unidentified, although as with most secondary metabolites they too most likely function in plant defense. Short-chain polyprenols may also be involved in protein glycosylation in plants, by analogy with the role of dolichols in animal metabolism.
The problem to be solved is to identify new plant genes having utility in plant defense mechanisms. Applicants have solved the stated problem by the identification of plant genes encoding plant cis-prenyltransferases. The present invention presents genes with significant homology to the bacterial UPP synthase and yeast Dedol-PP synthase from plants. The present invention shows that such genes are present in a range of plant species, including economically important crop plants such as cereals and the rubber tree Hevea brasiliensis, and thus are likely to be ubiquitous in plants.
This invention pertains to the identification and characterization of EST sequences from wheat, grape, soybean, rice, African daisy, rubber tree and pot marigold encoding cis-prenyltransferase proteins from these species.
It is an object of the present invention to provide an isolated nucleic acid fragment encoding a plant cis-prenyltransferase protein selected from the group consisting of: (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20; (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20; (c) an isolated nucleic acid fragment encoding a polypeptide, the polypeptide having at least 41% identity with the amino acid sequence set forth in SEQ ID NO:24; (d) an isolated nucleic acid fragment encoding having at least 50% identity with nucleic acid sequence as set forth in SEQ ID NO:23; (e) an isolated nucleic acid molecule that hybridizes with a nucleic acid sequence of (a) (b), (c) or (d) under the following hybridization conditions: 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 0.2xc3x97SSC, 0.5% SDS; (f) an isolated nucleic acid fragment that hybridizes with a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:19 under the following hybridization conditions 0.1xc3x97SSC, 0.1% SDS, 65xc2x0 C. and washed with 0.2xc3x97SSC, 0.5% SDS; and (g) an isolated nucleic acid fragment that is complementary to (a), (b), (c), (d), (e) or (f).
The invention further provides polypeptides encoded by the isolated nucleic acid fragments of the present invention, such as are presented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20.
In another embodiment the invention provides a chimeric gene comprising the isolated nucleic acid fragment of the present invention operably linked to suitable regulatory sequences.
The invention additionally provides a method of altering the level of expression of a plant cis-prenyltransferase protein in a host cell comprising: (a) transforming a host cell with the chimeric gene of the present invention and; (b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene resulting in production of altered levels of a plant cis-prenyltransferase protein in the transformed host cell relative to expression levels of an untransformed host cell. The invention further provides that where the cis-prenyltransferase protein is expressed in a transformed plant that the defense mechanism of the plant will be modulated.
The invention additionally provides transformed host cells comprising the chimeric genes of the present invention.
In an alternative embodiment the invention provides methods of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a plant cis-prenyltransferase protein using portions of the present nucleic acid sequences as hybridization probes or as primers.